Antisense modulation of focal adhesion kinase expression

ABSTRACT

Compounds, compositions and methods are provided for inhibiting FAK mediated signaling. The compositions comprise antisense compounds targeted to nucleic acids encoding FAK. Methods of using these antisense compounds for inhibition of FAK expression and for treatment of diseases, particularly cancers, associated with overexpression or constitutive activation of FAK are provided.

INTRODUCTION

[0001] This application is a continuation-in-part of the PCT ApplicationNo. PCT/US00/18999 filed Jul. 13, 2000 which corresponds to U.S.application Ser. No. 09/377,310 filed Aug. 19, 1999 now issued U.S. Pat.No. 6,133,031.

FIELD OF THE INVENTION

[0002] This invention relates to compositions and methods for modulatingexpression of the human focal adhesion kinase (FAK) gene, which encodesa signaling protein involved in growth factor response and cellmigration and is implicated in disease. This invention is also directedto methods for inhibiting FAK-mediated signal transduction; thesemethods can be used diagnostically or therapeutically. Furthermore, thisinvention is directed to treatment of conditions associated withexpression of the human FAK gene.

BACKGROUND OF THE INVENTION

[0003] Cell migration is fundamental to a variety of biologicalprocesses and can be induced by both integrin receptor-mediated signals(haptotaxis migration) and/or soluble growth factor-mediated signals(chemotaxis migration). Integrin receptor engagement activates focaladhesion kinase (FAK, also pp125FAK), a non-receptor protein-tyrosinekinase localized to cell substratum-extracellular matrix (ECM) contactsites that function as part of a cytoskeletal-associated network ofsignaling proteins (Schlaepfer, D. D., et al., Prog. Diophys. Mol.Biol., 1999, 71, 435-478). In adherent cells, FAK is often associatedwith integrins at focal adhesions (Schaller, M. D., et al., Proc. Natl.Acad. Sci. USA, 1992, 89, 5192-5196). Numerous other signaling proteins,including other protein tyrosine kinases are associated with FAK atthese regions. Phosphorylation of FAK results in activation of themitogen-activated protein kinase pathway. In addition, FAK regulatesactivation of phosphatidylinositol 3′-kinase which may serve to preventapoptosis. FAK has also been shown to be required for internalization ofbacteria mediated by invasin (Alrutz, M. A. and Isberg, R. R., Proc.Natl. Acad. Sci. USA, 1998, 95, 13658-13663).

[0004] Normal cells typically require anchorage to the extracellularmatrix in order to grow. When these cells are removed from theextracellular matrix, they undergo apoptosis. Transformed cells, on theother hand, can grow under anchorage-independent conditions, providingthem a growth advantage and the ability to be removed from their normalcellular environment.

[0005] Overexpression of FAK is involved in cancer progression. Highlevels of FAK correlates with invasiveness and metastatic potential incolon tumors (Weiner, T. M., et al., Lancet, 1993, 342, 1024-1025),breast tumors (Owens, L. V., et al., Cancer Res., 1995, 55, 2752-2755),and oral cancers (Kornberg, L. J., Head Neck, 1998, 20, 634-639).

[0006] FAK's role in cell migration has led to the speculation that itmay be relevant in other diseases such as embryonic developmentdisfunctions and angiogenic disorders (Kornberg, L. J., Head Neck, 1998,20, 634-639).

[0007] There is a lack of specific inhibitors of FAK. Antisenseapproaches have been a means by which the function of FAK has beeninvestigated. Lou, J. et al. (J. Orthopaedic Res., 1997, 15, 911-918)used an adenoviral based vector to express antisense FAK RNA to showthat FAK is involved in wound healing in tendons. Another antisense FAKexpression vector containing 400 bp of complementary sequence was usedto study the interaction of type I collagen and ?2?1 integrin (Takeuchi,Y., et al., J. Biol. Chem., 1997, 272, 29309-29316).

[0008] Antisense oligonucleotides have been used in several studies.Tanaka, S. et al. (J. Cell. Biochem., 1995, 58, 424-435) disclose twoantisense phosphorothioate oligonucleotides targeted to the start siteof mouse FAK. Xu, L. -H., et al. (Cell Growth Diff., 1996, 7, 413-418)disclose two antisense phosphorothioate oligonucleotides targeted withinthe coding region of human FAK. They also show that FAK antisensetreatment could induce apoptosis in tumor cells. Sonoda, Y., et al.(Biochem. Biophys. Res. Comm., 1997, 241, 769-774) also demonstrated arole for FAK in apoptosis using antisense phosphorothioateoligonucleotides targeted to the start site and within the coding regionof human FAK. Shibata, K., et al. (Cancer Res., 1998, 58, 900-903)disclose antisense phosphorothioate oligonucleotides targeted to thestart site and coding region of human FAK. Narase, K., et al. (Oncogene,1998, 17, 455-463) disclose an antisense phosphorothioateoligonucleotide targeted to the start site of human FAK.

[0009] There remains a long-felt need for improved compositions andmethods for inhibiting FAK gene expression.

SUMMARY OF THE INVENTION

[0010] The present invention provides antisense compounds which aretargeted to nucleic acids encoding focal adhesion kinase expression(FAK) and are capable of modulating FAK mediated signaling. The presentinvention also provides chimeric oligonucleotides targeted to nucleicacids encoding human FAK. The antisense compounds of the invention arebelieved to be useful both diagnostically and therapeutically, and arebelieved to be particularly useful in the methods of the presentinvention.

[0011] The present invention also comprises methods of modulating FAKmediated signaling, in cells and tissues, using the antisense compoundsof the invention. Methods of inhibiting FAK expression are provided;these methods are believed to be useful both therapeutically anddiagnostically. These methods are also useful as tools, for example, fordetecting and determining the role of FAK in various cell functions andphysiological processes and conditions and for diagnosing conditionsassociated with expression of FAK.

[0012] The present invention also comprises methods for diagnosing andtreating cancers, including those of the colon, breast and mouth. Thesemethods are believed to be useful, for example, in diagnosingFAK-associated disease progression. These methods employ the antisensecompounds of the invention. These methods are believed to be useful boththerapeutically, including prophylactically, and as clinical researchand diagnostic tools.

DETAILED DESCRIPTION OF THE INVENTION

[0013] FAK plays important roles in integrin-mediated signaltransduction. Overexpression of FAK is associated with tumor progressionand metastatic potential. As such, this protein represents an attractivetarget for treatment of such diseases. In particular, modulation of theexpression of FAK may be useful for the treatment of diseases such ascolon cancer, breast cancer and cancer of the mouth.

[0014] The present invention employs antisense compounds, particularlyoligonucleotides, for use in modulating the function of nucleic acidmolecules encoding FAK, ultimately modulating the amount of FAKproduced. This is accomplished by providing oligonucleotides whichspecifically hybridize with nucleic acids, preferably mRNA, encodingFAK.

[0015] This relationship between an antisense compound such as anoligonucleotide and its complementary nucleic acid target, to which ithybridizes, is commonly referred to as “antisense”. “Targeting” anoligonucleotide to a chosen nucleic acid target, in the context of thisinvention, is a multistep process. The process usually begins withidentifying a nucleic acid sequence whose function is to be modulated.This may be, as examples, a cellular gene (or MRNA made from the gene)whose expression is associated with a particular disease state, or aforeign nucleic acid from an infectious agent. In the present invention,the targets are nucleic acids encoding FAK; in other words, a geneencoding FAK, or mRNA expressed from the FAK gene. mRNA which encodesFAK is presently the preferred target. The targeting process alsoincludes determination of a site or sites within the nucleic acidsequence for the antisense interaction to occur such that modulation ofgene expression will result.

[0016] In accordance with this invention, persons of ordinary skill inthe art will understand that messenger RNA includes not only theinformation to encode a protein using the three letter genetic code, butalso associated ribonucleotides which form a region known to suchpersons as the 5′-untranslated region, the 3′-untranslated region, the5′ cap region and intron/exon junction ribonucleotides. Thus,oligonucleotides may be formulated in accordance with this inventionwhich are targeted wholly or in part to these associated ribonucleotidesas well as to the informational ribonucleotides. The oligonucleotide maytherefore be specifically hybridizable with a transcription initiationsite region, a translation initiation codon region, a 5′ cap region, anintron/exon junction, coding sequences, a translation termination codonregion or sequences in the 5′- or 3′-untranslated region. Since, as isknown in the art, the translation initiation codon is typically 5′-AUG(in transcribed MRNA molecules; 5′-ATG in the corresponding DNAmolecule), the translation initiation codon is also referred to as the“AUG codon,” the “start codon” or the “AUG start codon.” A minority ofgenes have a translation initiation codon having the RNA sequence5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shownto function in vivo. Thus, the terms “translation initiation codon” and“start codon” can encompass many codon sequences, even though theinitiator amino acid in each instance is typically methionine (ineukaryotes) or formylmethionine (prokaryotes). It is also known in theart that eukaryotic and prokaryotic genes may have two or morealternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAmolecule transcribed from a gene encoding FAK, regardless of thesequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms“start codon region,” “AUG region” and “translation initiation codonregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,5′ or 3′) from a translation initiation codon. This region is apreferred target region. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anMRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon. This region is a preferred target region. The openreading frame (ORF) or “coding region,” which is known in the art torefer to the region between the translation initiation codon and thetranslation termination codon, is also a region which may be targetedeffectively. Other preferred target regions include the 51 untranslatedregion (5′UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene andthe 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an MRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5¹-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0017] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns”, whichare excised from a pre-mRNA transcript to yield one or more mature mRNA.The remaining (and therefore translated) regions are known as “exons”and are spliced together to form a continuous mRNA sequence. mRNA splicesites, i.e., exon-exon or intron-exon junctions, may also be preferredtarget regions, and are particularly useful in situations where aberrantsplicing is implicated in disease, or where an overproduction of aparticular MRNA splice product is implicated in disease. Aberrant fusionjunctions due to rearrangements or deletions are also preferred targets.Targeting particular exons in alternatively spliced mRNAs may also bepreferred. It has also been found that introns can also be effective,and therefore preferred, target regions for antisense compoundstargeted, for example, to DNA or pre-mRNA.

[0018] Once the target site or sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired modulation.

[0019] “Hybridization”, in the context of this invention, means hydrogenbonding, also known as Watson-Crick base pairing, between complementarybases, usually on opposite nucleic acid strands or two regions of anucleic acid strand. Guanine and cytosine are examples of complementarybases which are known to form three hydrogen bonds between them. Adenineand thymine are examples of complementary bases which form two hydrogenbonds between them.

[0020] “Specifically hybridizable” and “complementary” are terms whichare used to indicate a sufficient degree of complementarity such thatstable and specific binding occurs between the DNA or RNA target and theoligonucleotide.

[0021] It is understood that an oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.

[0022] Hybridization of antisense oligonucleotides with mRNA interfereswith one or more of the normal functions of MRNA. The functions of mRNAto be interfered with include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA.Binding of specific protein(s) to the RNA may also be interfered with byantisense oligonucleotide hybridization to the RNA.

[0023] The overall effect of interference with MRNA function ismodulation of expression of FAK. In the context of this invention“modulation” means either inhibition or stimulation; i.e., either adecrease or increase in expression. This modulation can be measured inways which are routine in the art, for example by Northern blot assay ofmRNA expression, or reverse transcriptase PCR, as taught in the examplesof the instant application or by Western blot or ELISA assay of proteinexpression, or by an immunoprecipitation assay of protein expression.Effects on cell proliferation or tumor cell growth can also be measured,as taught in the examples of the instant application. Inhibition ispresently preferred.

[0024] The oligonucleotides of this invention can be used indiagnostics, therapeutics, prophylaxis, and as research reagents and inkits. Since the oligonucleotides of this invention hybridize to nucleicacids encoding FAK, sandwich, calorimetric and other assays can easilybe constructed to exploit this fact. Provision of means for detectinghybridization of oligonucleotide with the FAK genes or mRNA canroutinely be accomplished. Such provision may include enzymeconjugation, radiolabelling or any other suitable detection systems.Kits for detecting the presence or absence of FAK may also be prepared.

[0025] The present invention is also suitable for diagnosing abnormalinflammatory states or certain cancers in tissue or other samples frompatients suspected of having an autoimmune or inflammatory disease suchas hepatitis or cancers such as those of the colon, liver or lung, andlymphomas. A number of assays may be formulated employing the presentinvention, which assays will commonly comprise contacting a tissuesample with an oligonucleotide of the invention under conditionsselected to permit detection and, usually, quantitation of suchinhibition. In the context of this invention, to “contact” tissues orcells with an oligonucleotide or oligonucleotides means to add theoligonucleotide(s), usually in a liquid carrier, to a cell suspension ortissue sample, either in vitro or ex vivo, or to administer theoligonucleotide(s) to cells or tissues within an animal.

[0026] The oligonucleotides of this invention may also be used forresearch purposes. Thus, the specific hybridization exhibited by theoligonucleotides may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

[0027] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleicacid. This term includes s-s oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent intersugar(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced binding to target and increased stability in thepresence of nucleases.

[0028] The antisense compounds in accordance with this inventionpreferably comprise from about 5 to about 50 nucleobases. Particularlypreferred are antisense oligonucleotides comprising from about 8 toabout 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides).As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2=, 3= or 5=hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3= to 5=phosphodiesterlinkage.

[0029] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0030] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3=-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3=-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3=-5=linkages, 2=-5=linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3=-5=to 5=-3= or 2=-5= to 5=-2=. Various salts, mixed salts and free acidforms are also included.

[0031] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; and 5,625,050.

[0032] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

[0033] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439.

[0034] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNAcompounds can be found in Nielsen et al. (Science, 1991, 254,1497-1500).

[0035] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0036] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O-, S-, or N-alkyl,O-alkyl-O-alkyl, O-, S-, or N-alkenyl, or O-, S- or N-alkynyl, whereinthe alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ toC₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. Other preferred oligonucleotides compriseone of the following at the 2= position: C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and2′-dimethylamino-ethoxyethoxy (2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)2.

[0037] Other preferred modifications include 2¹-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2=-5= linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative U.S. patents that teach the preparation of suchmodified sugars structures include, but are not limited to, U.S. Pat.Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873;5,670,633; and 5,700,920.

[0038] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C orm5c), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in the Concise Encyclopedia Of PolymerScience And Engineering 1990, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, those disclosed by Englisch et al. (Angewandte Chemie,International Edition 1991, 30, 613-722), and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications 1993,pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications 1993, CRC Press, Boca Raton, pages 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

[0039] Representative U.S. patents that teach the preparation of certainof the above noted modified nucleobases as well as other modifiednucleobases include, but are not limited to, the above noted U.S. Pat.No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941.

[0040] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Lett. 1994, 4, 1053-1059), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci. 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let. 1993,3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J. 1991, 10, 1111-1118; Kabanovet al., FEBS Lett. 1990, 259, 327-330; Svinarchuk et al., Biochimie1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res. 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett.1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al ., J. Pharmacol.Exp. Ther. 1996, 277, 923-937).

[0041] Representative U.S. patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0042] The present invention also includes oligonucleotides which arechimeric oligonucleotides. “Chimeric” oligonucleotides or “chimeras,” inthe context of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionwherein the oligonucleotide is modified so as to confer upon theoligonucleotide increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof antisense inhibition of gene expression. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art. ThisRNAse H-mediated cleavage of the RNA target is distinct from the use ofribozymes to cleave nucleic acids. Ribozymes are not comprehended by thepresent invention.

[0043] Examples of chimeric oligonucleotides include but are not limitedto “gapmers,” in which three distinct regions are present, normally witha central region flanked by two regions which are chemically equivalentto each other but distinct from the gap. A preferred example of a gapmeris an oligonucleotide in which a central portion (the “gap”) of theoligonucleotide serves as a substrate for RNase H and is preferablycomposed of 2′-deoxynucleotides, while the flanking portions (the 5′ and3′ “wings”) are modified to have greater affinity for the target RNAmolecule but are unable to support nuclease activity (e.g., fluoro- or21—O-methoxyethyl-substituted). Chimeric oligonucleotides are notlimited to those with modifications on the sugar, but may also includeoligonucleosides or oligonucleotides with modified backbones, e.g., withregions of phosphorothioate (P═S) and phosphodiester (P═O) backbonelinkages or with regions of MMI and P═S backbone linkages. Otherchimeras include “wingmers,” also known in the art as “hemimers,” thatis, oligonucleotides with two distinct regions. In a preferred exampleof a wingmer, the 5′ portion of the oligonucleotide serves as asubstrate for RNase H and is preferably composed of 2′-deoxynucleotides,whereas the 3′ portion is modified in such a fashion so as to havegreater affinity for the target RNA molecule but is unable to supportnuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl- substituted),or vice-versa. In one embodiment, the oligonucleotides of the presentinvention contain a 2′-O-methoxyethyl (2′-O—CH₂CH₂O CH₃) modification onthe sugar moiety of at least one nucleotide. This modification has beenshown to increase both affinity of the oligonucleotide for its targetand nuclease resistance of the oligonucleotide. According to theinvention, one, a plurality, or all of the nucleotide subunits of theoligonucleotides of the invention may bear a 2′-O-methoxyethyl(—O—CH₂CH₂OCH₃) modification. Oligonucleotides comprising a plurality ofnucleotide subunits having a 2′-O-methoxyethyl modification can havesuch a modification on any of the nucleotide subunits within theoligonucleotide, and may be chimeric oligonucleotides. Aside from or inaddition to 2′-O-methoxyethyl modifications, oligonucleotides containingother modifications which enhance antisense efficacy, potency or targetaffinity are also preferred. Chimeric oligonucleotides comprising one ormore such modifications are presently preferred.

[0044] The oligonucleotides used in accordance with this invention maybe conveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and 2′-alkoxy or 2¹-alkoxyalkoxy derivatives,including 2′-Q-methoxyethyl oligonucleotides (Martin, P., Helv. Chim.Acta 1995, 78, 486-504). It is also well known to use similar techniquesand commercially available modified amidites and controlled-pore glass(CPG) products such as biotin, fluorescein, acridine orpsoralen-modified amidites and/or CPG (available from Glen Research,Sterling, VA) to synthesize fluorescently labeled, biotinylated or otherconjugated oligonucleotides.

[0045] The antisense compounds of the present invention includebioequivalent compounds, including pharmaceutically acceptable salts andprodrugs. This is intended to encompass any pharmaceutically acceptablesalts, esters, or salts of such esters, or any other compound which,upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto pharmaceutically acceptable salts of the nucleic acids of theinvention and prodrugs of such nucleic acids. Pharmaceuticallyacceptable salts are physiologically and pharmaceutically acceptablesalts of the nucleic acids of the invention: i.e., salts that retain thedesired biological activity of the parent compound and do not impartundesired toxicological effects thereto (see, for example, Berge et al.,“Pharmaceutical Salts,” J. of Pharma Sci. 1977, 66, 1-19).

[0046] For oligonucleotides, examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0047] The oligonucleotides of the invention may additionally oralternatively be prepared to be delivered in a Aprodrug form. The termAprodrug indicates a therapeutic agent that is prepared in an inactiveform that is converted to an active form (i.e., drug) within the body orcells thereof by the action of endogenous enzymes or other chemicalsand/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published December 9, 1993.

[0048] For therapeutic or prophylactic treatment, oligonucleotides areadministered in accordance with this invention. Oligonucleotidecompounds of the invention may be formulated in a pharmaceuticalcomposition, which may include pharmaceutically acceptable carriers,thickeners, diluents, buffers, preservatives, surface active agents,neutral or cationic lipids, lipid complexes, liposomes, penetrationenhancers, carrier compounds and other pharmaceutically acceptablecarriers or excipients and the like in addition to the oligonucleotide.Such compositions and formulations are comprehended by the presentinvention.

[0049] Pharmaceutical compositions comprising the oligonucleotides ofthe present invention may include penetration enhancers in order toenhance the alimentary delivery of the oligonucleotides. Penetrationenhancers may be classified as belonging to one of five broadcategories, i.e., fatty acids, bile salts, chelating agents, surfactantsand non-surfactants (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems 1991, 8, 91-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems 1990, 7, 1-33). One or more penetrationenhancers from one or more of these broad categories may be included.

[0050] Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid,linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems 1990, 7, 1; El-Hariri et al., J. Pharm.Pharmacol. 1992 44, 651-654).

[0051] The physiological roles of bile include the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York,N.Y., 1996, pages 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus, the term“bile salt” includes any of the naturally occurring components of bileas well as any of their synthetic derivatives.

[0052] Complex formulations comprising one or more penetration enhancersmay be used. For example, bile salts may be used in combination withfatty acids to make complex formulations.

[0053] Chelating agents include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)[Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems 1990, 7, 1-33; Buur et al., J. ControlRel. 1990, 14, 43-51). Chelating agents have the added advantage of alsoserving as DNase inhibitors.

[0054] Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al.,J. Pharm. Phamacol. 1988, 40, 252-257).

[0055] Non-surfactants include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92); andnon-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.1987, 39, 621-626).

[0056] As used herein, “carrier compound” refers to a nucleic acid, oranalog thereof, which is inert (i.e., does not possess biologicalactivity per se) but is recognized as a nucleic acid by in vivoprocesses that reduce the bioavailability of a nucleic acid havingbiological activity by, for example, degrading the biologically activenucleic acid or promoting its removal from circulation. Thecoadministration of a nucleic acid and a carrier compound, typicallywith an excess of the latter substance, can result in a substantialreduction of the amount of nucleic acid recovered in the liver, kidneyor other extracirculatory reservoirs, presumably due to competitionbetween the carrier compound and the nucleic acid for a common receptor.

[0057] In contrast to a carrier compound, a “pharmaceutically acceptablecarrier” (excipient) is a pharmaceutically acceptable solvent,suspending agent or any other pharmacologically inert vehicle fordelivering one or more nucleic acids to an animal. The pharmaceuticallyacceptable carrier may be liquid or solid and is selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, etc., when combined with a nucleic acid andthe other components of a given pharmaceutical composition. Typicalpharmaceutically acceptable carriers include, but are not limited to,binding agents (e.g., pregelatinized maize starch, polyvinyl-pyrrolidoneor hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate,etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidalsilicon dioxide, stearic acid, metallic stearates, hydrogenatedvegetable oils, corn starch, polyethylene glycols, sodium benzoate,sodium acetate, etc.); disintegrates (e.g., starch, sodium starchglycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate,etc.). Sustained release oral delivery systems and/or enteric coatingsfor orally administered dosage forms are described in U.S. Pat. Nos.4,704,295; 4,556,552; 4,309,406; and 4,309,404.

[0058] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional compatiblepharmaceutically-active materials such as, e.g. , antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the composition of present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the invention.

[0059] Regardless of the method by which the oligonucleotides of theinvention are introduced into a patient, colloidal dispersion systemsmay be used as delivery vehicles to enhance the in vivo stability of theoligonucleotides and/or to target the oligonucleotides to a particularorgan, tissue or cell type. Colloidal dispersion systems include, butare not limited to, macromolecule complexes, nanocapsules, microspheres,beads and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, liposomes and lipid:oligonucleotide complexesof uncharacterized structure. A preferred colloidal dispersion system isa plurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layers made up of lipidsarranged in a bilayer configuration (see, generally, Chonn et al.,Current Op. Biotech. 1995, 6, 698-708).

[0060] The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, epidermal, and transdermal), oral or parenteral. Parenteraladministration includes intravenous drip, subcutaneous, intraperitonealor intramuscular injection, pulmonary administration, e.g., byinhalation or insufflation, or intracranial, e.g., intrathecal orintraventricular, administration. Oligonucleotides with at least one2′-O-methoxyethyl modification are believed to be particularly usefulfor oral administration.

[0061] Formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

[0062] Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

[0063] Compositions for parenteral administration may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives. In some cases it may be more effective to treat apatient with an oligonucleotide of the invention in conjunction withother traditional therapeutic modalities in order to increase theefficacy of a treatment regimen. In the context of the invention, theterm “treatment regimen” is meant to encompass therapeutic, palliativeand prophylactic modalities. For example, a patient may be treated withconventional chemotherapeutic agents, particularly those used for tumorand cancer treatment. Examples of such chemotherapeutic agents includebut are not limited to daunorubicin, daunomycin, dactinomycin,doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide,ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,mitomycin C, actinomycin D, mithramycin, prednisone,hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al.,eds., Rahway, N.J. When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).

[0064] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in vitro andin in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0065] The following examples illustrate the present invention and arenot intended to limit the same.

EXAMPLES Example 1

[0066] Synthesis of Oligonucleotides

[0067] Unmodified oligodeoxynucleotides are synthesized on an automatedDNA synthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.S-cyanoethyldiisopropyl-phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of³H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step. Cytosinesmay be 5-methyl cytosines. (5-methyl deoxycytidine phosphoramiditesavailable from Glen Research, Sterling, Va. or Amersham PharmaciaBiotech, Piscataway, N.J.) 2′-methoxy oligonucleotides are synthesizedusing 2′-methoxy 9-cyanoethyldiisopropyl-phosphoramidites (Chemgenes,Needham, Mass.) and the standard cycle for unmodified oligonucleotides,except the wait step after pulse delivery of tetrazole and base isincreased to 360 seconds. Other 2′-alkoxy oligonucleotides aresynthesized by a modification of this method, using appropriate2′-modified amidites such as those available from Glen Research, Inc.,Sterling, Va. 2′-fluoro oligonucleotides are synthesized as described inKawasaki et al. (J. Med. Chem. 1993, 36, 831-841). Briefly, theprotected nucleoside N⁶-benzoyl-2′-deoxy-2′-fluoroadenosine issynthesized utilizing commercially available9-β-D-arabinofuranosyladenine as starting material and by modifyingliterature procedures whereby the 2′-a-fluoro atom is introduced by aS_(N)2-displacement of a 2′-β-O-trifyl group. ThusN⁶-benzoyl-9-β-D-arabinofuranosyladenine is selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N⁶-benzoyl groups is accomplished usingstandard methodologies and standard methods are used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0068] The synthesis of 2¹-deoxy-2′-fluoroguanosine is accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-β-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyrylarabinofuranosylguanosine. Deprotection ofthe TPDS group is followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation is followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies are used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0069] Synthesis of 2′-deoxy-2′-fluorouridine is accomplished by themodification of a known procedure in which 2,2′-anhydro-1-β-D-arabinofuranosyluracil is treated with 70% hydrogenfluoride-pyridine. Standard procedures are used to obtain the 5¹-DMT and5′-DMT-3′phosphoramidites.

[0070] 2¹-deoxy-2¹-fluorocytidine is synthesized via amination of2¹-deoxy-2¹-fluorouridine, followed by selective protection to giveN⁴-benzoyl-2¹-deoxy-2′-fluorocytidine. Standard procedures are used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0071] 2′-(2-methoxyethyl)-modified amidites were synthesized accordingto Martin, P. (Helv. Chim. Acta 1995, 78, 486-506). For ease ofsynthesis, the last nucleotide may be a deoxynucleotide.2-O—CH₂CH₂OCH₃-cytosines may be 5-methyl cytosines.

[0072] Synthesis of 5-Methyl cytosine monomers:

[0073] 2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]:

[0074] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 h) to give a solid which was crushed to a light tan powder(57 g, 85% crude yield). The material was used as is for furtherreactions.

[0075] 2′-O-Methoxyethyl-5-methyluridine:

[0076]

[0077] 2,2′-Anhydro-5-methyluridine (195 9, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 9) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product.

[0078] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine:

[0079] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄₁ filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/Hexane/Acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 9of product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0080]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine:

[0081] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by tic by first quenching the tic sample with the additionof MeOH. Upon completion of the reaction, as judged by tic, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%).3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine:

[0082] A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) inCH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was addedto a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L), cooled to -5?Cand stirred for 0.5 h using an overhead stirrer. POCl₃ was addeddropwise, over a 30 minute period, to the stirred solution maintained at0-10° C., and the resulting mixture stirred for an additional 2 hours.The first solution was added dropwise, over a 45 minute period, to thelater solution. The resulting reaction mixture was stored overnight in acold room. Salts were filtered from the reaction mixture and thesolution was evaporated. The residue was dissolved in EtOAc (1 L) andthe insoluble solids were removed by filtration. The filtrate was washedwith 1×300 mL of NaHCO₃ and 2×300 mL of saturated NaCl, dried oversodium sulfate and evaporated. The residue was triturated with EtOAc togive the title compound.

[0083] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:

[0084] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-0-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄0H (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0085]N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:

[0086] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, tlc showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/Hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0087]N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite:

[0088]N⁴-Benzoyl-21—O-methoxyethyl-51—O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAcHexane (3:1) as theeluting solvent. The pure fractions were combined to give 90.6 g (87%)of the title compound.

[0089] 5-methyl-21-deoxycytidine (5-me-C) containing oligonucleotideswere synthesized according to published methods (Sanghvi et al., Nucl.Acids Res. 1993, 21, 3197-3203) using commercially availablephosphoramidites (Glen Research, Sterling Va. or ChemGenes, NeedhamMass.).

[0090] 2=-O-(dimethylaminooxyethyl) nucleoside amidites

[0091] 2¹-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2¹—O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0092] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0093] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.09, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

[0094]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0095] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 9, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

[0096] 2-O—-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0097]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethylazodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819, 86%).

[0098] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0099]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 hr the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eg.) was addedand the mixture for 1 hr. Solvent was removed under vacuum; residuechromatographed to get5-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95, 78%).

[0100]5′-O-tert-Butyldiphenylsilyl-2-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0101]5-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 hr, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 100C for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO, and evaporated to dryness . The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).2′-O-(dimethylaminooxyethyl)-5-methyluridine Triethylaminetrihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF andtriethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture oftriethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5 -methyluridine (766 mg,92.5%).

[0102] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3,-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0103] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N,N-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol)was added. The reaction mixture was stirred at ambient temperature for 4hrs under inert atmosphere. The progress of the reaction was monitoredby TLC (hexane:ethyl acetate 1:1). The solvent was evaporated, then theresidue was dissolved in ethyl acetate (70 mL) and washed with 5%aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as afoam (1.04 g, 74.9%).

[0104] Oligonucleotides having methylene (methylimino) (MMI) backbonesare synthesized according to U.S. Pat. No. 5,378,825, which iscoassigned to the assignee of the present invention and is incorporatedherein in its entirety. For ease of synthesis, various nucleoside dimerscontaining MMI linkages are synthesized and incorporated intooligonucleotides. Other nitrogen-containing backbones are synthesizedaccording to WO 92/20823 which is also coassigned to the assignee of thepresent invention and incorporated herein in its entirety.

[0105] Oligonucleotides having amide backbones are synthesized accordingto De Mesmaeker et al. (Acc. Chem. Res. 1995, 28, 366-374). The amidemoiety is readily accessible by simple and well-known synthetic methodsand is compatible with the conditions required for solid phase synthesisof oligonucleotides.

[0106] Oligonucleotides with morpholino backbones are synthesizedaccording to U.S. Pat. No. 5,034,506 (Summerton and Weller).

[0107] Peptide-nucleic acid (PNA) oligomers are synthesized according toP. E. Nielsen et al. (Science 1991, 254, 1497-1500).

[0108] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides are purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotideswere analyzed by polyacrylamide gel electrophoresis on denaturing gelsor capillary gel electrophoresis and judged to be at least 85% fulllength material. The relative amounts of -phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹p nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.(J. Biol. Chem. 1991, 266, 18162). Results obtained with HPLC-purifiedmaterial were similar to those obtained with non-HPLC purified material.

[0109] Alternatively, oligonucleotides are synthesized in 96 well plateformat via solid phase P(III) phosphoramidite chemistry on an automatedsynthesizer capable of assembling 96 sequences simultaneously in astandard 96 well format. Phosphodiester internucleotide linkages areafforded by oxidation with aqueous iodine. Phosphorothioateinternucleotide linkages are generated by sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrousacetonitrile. Standard base-protected beta-cyanoethyl-di-isopropylphosphoramidites are purchased from commercial vendors (e.g. PE-AppliedBiosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.).Non-standard nucleosides are synthesized as per published methods. Theyare utilized as base protected beta-cyanoethyldiisopropylphosphoramidites.

[0110] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 2

[0111] Human FAK Oligonucleotide Sequences Antisense oligonucleotideswere designed to target human FAK. Target sequence data are from thefocal adhesion kinase (FAK) cDNA sequence published by Whitney, G. S.,et al. (DNA Cell Biol., 1993, 12, 823-830); Genbank accession numberL13616, provided herein as SEQ ID NO: 1. One set of oligonucleotideswere synthesized as chimeric oligonucleotides (“gapmers”), 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings.” The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P=S) throughout the oligonucleotide. All2′-MOE cytosines were 5-methylcytosines. These oligonucleotide sequencesare shown in Table 1. An identical set of sequences were prepared asfully phosphorothioated oligodeoxynucleotides. These are shown in Table2. An additional set of oligonucleotides were synthesized as chimericoligonucleotides (“gapmers”), 15 nucleotides in length, composed of acentral “gap” region consisting of five 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.”The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P=S)throughout the oligonucleotide. All 2′-MOE cytosines were5-methyl-cytosines. These oligonucleotide sequences are shown in Table3. An identical set of sequences were prepared as fullyphosphorothioated oligodeoxynucleotides. These are shown in Table 4.

[0112] Human A549 lung carcinoma cells (American Type CultureCollection, Manassas, Va.) were grown in DMEM supplemented with 10%fetal bovine serum (FBS), non-essential amino acids for MEM, sodiumpyruvate (1 mM), penicillin (50 U/ml) and streptomycin (50 μg/ml). Allcell culture reagents were obtained from Life Technologies (Rockville,Md.).

[0113] The cells were washed once with OPTIMEM™ (Life Technologies,Rockville, Md.), then transfected with 400 nM oligonucleotide and 12mg/ml LIPOFECTIN^(R) (Life Technologies, Rockville, MD), a 1:1 (w/w)liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA),and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water.The cells were incubated with oligonucleotide for four hours, afterwhich the media was replaced with fresh media and the cells incubatedfor another 20 hours.

[0114] Total cellular RNA was isolated using an ATLAS™ Pure RNAisolation kit (Clontech, Palo Alto, Calif.). RNA was then separated on a1.2% agarose-formaldehyde gel, transferred to Hybond-N+membrane(Amersham Pharmacia Biotech, Arlington Heights, Ill.), a positivelycharged nylon membrane. Immobilized RNA was cross-linked by exposure toUV light. Membranes were probed with either FAK or glyceraldehyde3-phosphate dehydrogenase (G3PDH) probes. The probes were labeled byrandom primer using the PRIME-A-GENE⁷ Labeling System, Promega, Madison,Wis.) and hybridized to the membranes. mRNA signals were quantitated bya PhosphoImager (Molecular Dynamics, Sunnyvale, Calif.).

[0115] Results of an initial screen of the FAK antisenseoligonucleotides are shown in Tables 5 (20 mers) and 6 (15 ers).Oligonucleotides 15392 (SEQ ID NO. 3), 15394 (SEQ ID O. 4), 15397 (SEQID NO. 6), 15399 (SEQ ID NO. 7), 15401 (SEQ ID NO. 8), 15403 (SEQ ID NO.9), 15405 (SEQ ID NO. 10), 15407 (SEQ ID NO. 11), 15409 (SEQ ID NO. 12),15413 (SEQ ID NO. 14), 15415 (SEQ ID NO. 15), 15458 (SEQ ID NO. 16),15460 (SEQ ID NO. 17), 15421 (SEQ ID NO. 18), 15425 (SEQ ID NO. 20),15393 (SEQ ID NO. 23), 15406 (SEQ ID NO. 30), 15408 (SEQ ID NO. 31) and15412 (SEQ ID NO. 33) resulted in about 50% or greater inhibition of FAKmRNA expression in this assay. Oligonucleotides 15401 (SEQ ID NO. 8),15403 (SEQ ID NO. 9), 15409 (SEQ ID NO. 12), 15413 (SEQ ID NO. 14),15415 (SEQ ID NO. 15), and 15421 (SEQ ID NO. 18) resulted in about 80%or greater inhibition of FAK mRNA expression. TABLE 1 NucleotideSequences of Human FAK Chimeric (deoxy gapped) 20 mer PhosphorothioateOligonucleotides NUCLEOTIDE SEQ TARGET GENE GENE ISIS SEQUENCE¹ IDNUCLEOTIDE TARGET NO. (5′ → 3′) NO: CO-ORDINATES² REGION 15392CCGCGGGCTCACA  3 0001-0020 5′-UTR GTGGTCG 15394 GGCGCCGTGAAGC  40078-0097 5′-UTR GAAGGCA 15395 CAGTTCTGCTCGG  5 0101-0120 5′-UTR ACCGCGG15397 GAAACTGCAGAAG  6 0150-0169 5′-UTR GCACTGA 15399 TTCTCCCTTCCGT  70183-0202 5′-UTR TATTCTT 15401 CTAGATGCTAGGT  8 0206-0225 5′-UTR ATCTGTC15403 TTTTGCTAGATGC  9 0211-0230 5′-UTR TAGGTAT 15405 GGTAAGCAGCTGC 100229-0248 start CATTATT 15407 AGTACCCAGGTGA 11 0285-0304 coding GTCTTAG15409 CCTGACATCAGTA 12 0408-0427 coding GCATCTC 15411 GTTGGCTTATCTT 130641-0660 coding CAGTAAA 15413 GGTTAGGGATGGT 14 1218-1237 coding GCCGTCA15415 TGTTCGTTTCCAA 15 2789-2808 coding TCGGACC 15417 CTAGGGGAGGCTC 163383-3402 stop AGTGTGG 15419 ATTCCTCGCTGCT 17 3444-3463 3′-UTR GGTGGAA15421 TTTCAACCAGATG 18 3510-3529 3′-UTR GTCATTC 15423 TTCTGAATATCAT 193590-3609 3′-UTR GATTGAA 15425 CATGATGCTTAAA 20 3658-3677 3′-UTR AGCTTAC15427 AATGTGAACATAA 21 3680-3699 3′-UTR ATTGTTC 15429 AAGGTAGTTTAGG 223738-3757 3′-UTR AATTAAG

[0116] TABLE 2 Nucleotide Sequences of Human FAK 20 mer PhosphorothioateOligonucleotides NUCLEOTIDE SEQ TARGET GENE GENE ISIS SEQUENCE¹ IDNUCLEOTIDE TARGET NO. (5′ → 3′) NO: CO-ORDINATES² REGION 15432CCGCGGGCTCACA  3 0001-0020 5′-UTR GTGGTCG 15434 GGCGCCGTGAAGC  40078-0097 5′-UTR GAAGGCA 15436 CAGTTCTGCTCGG  5 0101-0120 5′-UTR ACCGCGG15438 GAAACTGCAGAAG  6 0150-0169 5′-UTR GCACTGA 15440 TTCTCCCTTCCGT  70183-0202 5′-UTR TATTCTT 15442 CTAGATGCTAGGT  8 0206-0225 5′-UTR ATCTGTC15444 TTTTGCTAGATGC  9 0211-0230 5′-UTR TAGCTAT 15446 GGTAAGCAGCTGC 100229-0248 start CATTATT 15448 AGTACCCAGGTGA 11 0285-0304 coding GTCTTAG15450 CCTCACATCAGTA 12 0408-0427 coding GCATCTC 15452 GTTGGCTTATCTT 130641-0660 coding CAGTAAA 15454 GGTTAGGGATGGT 14 1218-1237 coding GCCGTCA15456 TGTTGGTTTCCAA 15 2789-2808 coding TCGGACC 15458 CTAGCGGAGGCTC 163383-3402 stop AGTGTGG 15460 ATTCCTCGCTGCT 17 3444-3463 3′-UTR GGTGGAA15462 TTTCAACCAGATG 18 3510-3529 3′-UTR GTCATTC 15464 TTCTGAATATCAT 193590-3609 3′-UTR GATTGAA 15466 CATGATGCTTAAA 20 3658-3677 3′-UTR AGCTTAC15468 AATGTGAACATAA 21 3680-3699 3′-UTR ATTGTTC 15470 AAGGTAGTTTAGG 223738-3757 3′-UTR AATTAAG

[0117] TABLE 3 Nucleotide Sequences of Human FAK Chimeric (deoxy gapped)15 mer Phosphorothioate Oligonucleotides NUCLEOTIDE SEQ TARGET GENE GENEISIS SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ → 3′) NO: CO-ORDINATES²REGION 15393 GCGGGCTCACAGT 23 0004-0018 5′-UTR GG 15431 CGCCGTGAAGCGA 240081-0095 5′-UTR AG 15396 GTTCTGCTCGGAC 25 0104-0118 5′-UTR GC 15398AACTGCAGAAGGC 26 0153-0167 5′-UTR AC 15400 CTCCCTTCCGTTA 27 0186-02005′-UTR TT 15402 AGATGCTAGGTAT 28 0209-0223 5′-UTR CT 15404 TTGCTAGATGCTA29 0214-0228 5′-UTR GG 15406 TAAGCAGCTGCCA 30 0232-0246 start TT 15408TACCCAGGTGAGT 31 0288-0302 coding CT 15410 TGACATCAGTAGC 32 0411-0425coding AT 15412 TGGCTTATCTTCA 33 0644-0658 coding GT 15414 TTAGGGATGGTGC34 1221-1235 coding CG 15416 TTGGTTTCCAATC 35 2792-2806 coding GG 15418AGGGGAGGCTCAG 36 3386-3400 stop TG 15420 TCCTCGCTGCTGG 37 3447-34613′-UTR TG 15422 TCAACCAGATGGT 38 3513-3527 3′-UTR CA 15424 CTGAATATCATGA39 3593-3607 3′-UTR TT 15426 TGATGCTTAAAAG 40 3661-3675 3′-UTR CT 15428TGTGAACATAAAT 41 3683-3697 3′-UTR TG 15430 GGTAGTTTAGGAA 42 3741-37553′-UTR TT

[0118] TABLE 4 Nucleotide Sequences of Human FAK 15 mer PhosphorothioateOligonucleotides NUCLEOTIDE SEQ TARGET GENE GENE ISIS SEQUENCE¹ IDNUCLEOTIDE TARGET NO. (5′ → 3′) NO: CO-ORDINATES² REGION 15433GCGGGCTCACAGT 23 0004-0018 5′-UTR GG 15435 CGCCGTGAAGCGA 24 0081-00955′-UTR AG 15437 GTTCTGCTCGGAC 25 0104-0118 5′-UTR CG 15439 AACTGCAGAAGGC26 0153-0167 5′-UTR AC 15441 CTCCCTTCCGTTA 27 0186-0200 5′-UTR TT 15443AGATGCTAGGTAT 28 0209-0223 5′-UTR CT 15445 TTGCTAGATGCTA 29 0214-02285′-UTR GG 15447 TAAGCAGCTGCCA 30 0232-0246 start TT 15449 TACCCAGGTGAGT31 0288-0302 coding CT 15451 TGACATCAGTAGC 32 0411-0425 coding AT 15453TGGCTTATCTTCA 33 0644-0658 coding GT 15455 TTAGGGATGGTGC 34 1221-1235coding CG 15457 TTGGTTTCCAATC 35 2792-2806 coding GG 15459 AGGGGAGGCTCAG36 3386-3400 stop TG 15461 TCCTCGCTGCTGG 37 3447-3461 3′-UTR TG 15463TCAACCAGATGGT 38 3513-3527 3′-UTR CA 15465 CTGAATATCATGA 39 3593-36073′-UTR TT 15467 TGATGCTTAAAAG 40 3661-3675 3′-UTR CT 15469 TGTGAACATAAAT41 3683-3697 3′-UTR TG 15471 GGTAGTTTAGGAA 42 3741-3755 3′-UTR TT

[0119] TABLE 5 Inhibition of Human Fak mRNA expression in A549 Cells byFAK 20 mer Antisense Oligonucleotides SEQ GENE ISIS ID TARGET % mRNA %mRNA No: NO: REGION EXPRESSION INHIBITION control — — 100%  0% 15392  35′-UTR  29% 71% 15432  3 5′-UTR 108% — 15394  4 5′-UTR  30% 70% 15434  45′-UTR 147% — 15395  5 5′-UTR  57% 43% 15436  5 5′-UTR  88% 12% 15397  65′-UTR  31% 69% 15438  6 5′-UTR  64% 36% 15399  7 5′-UTR  48% 52% 15440 7 5′-UTR  92%  8% 15401  8 5′-UTR  17% 83% 15442  8 5′-UTR  63% 37%15403  9 5′-UTR  17% 83% 15444  9 5′-UTR 111% — 15405 10 start  46% 54%15446 10 start 145% — 15407 11 coding  36% 64% 15448 11 coding  90% 10%15409 12 coding  13% 87% 15450 12 coding 149% — 15411 13 coding  70% 30%15452 13 coding 129% — 15413 14 coding  22% 78% 15454 14 coding  82% 18%15415 15 coding  20% 80% 15456 15 coding  88% 12% 15417 16 stop  56% 44%15458 16 stop  39% 61% 15419 17 3′-UTR  55% 45% 15460 17 3′-UTR  42% 58%15421 18 3′-UTR  20% 80% 15462 18 3′-UTR  60% 40% 15423 19 3′-UTR  55%45% 15464 19 3′-UTR  97%  3% 15425 20 3′-UTR  51% 49% 15466 20 3′-UTR 74% 26% 15427 21 3′-UTR  67% 33% 15468 21 3′-UTR 131% — 15429 22 3′-UTR 57% 43% 15470 22 3′-UTR  71% 29%

[0120] TABLE 6 Inhibition of Human Fak mRNA expression in A549 Cells byFAK 15 mer antisense oligonucleotides SEQ GENE ISIS ID TARGET % mRNA %mRNA No: NO: REGION EXPRESSION INHIBITION control — — 100%  0% 15393 235′-UTR  40% 60% 15433 23 5′-UTR 160% — 15431 24 5′-UTR  59% 41% 15435 245′-UTR 121% — 15396 25 5′-UTR  76% 24% 15437 25 5′-UTR 123% — 15398 265′-UTR  72% 28% 15439 26 5′-UTR  64% 36% 15400 27 5′-UTR  79% 21% 1544127 5′-UTR  66% 34% 15402 28 5′-UTR  69% 31% 15443 28 5′-UTR  99%  1%15404 29 5′-UTR  70% 30% 15445 29 5′-UTR 151% — 15406 30 start  32% 68%15447 30 start  69% 31% 15408 31 coding  35% 65% 15449 31 coding  89%11% 15410 32 coding  67% 33% 15451 32 coding 142% — 15412 33 coding  43%57% 15453 33 coding 115% — 15414 34 coding  64% 36% 15455 34 coding  59%41% 15416 35 coding  69% 31% 15457 35 coding 121% — 15418 36 stop 140% —15459 36 stop  72% 28% 15420 37 3′-UTR 158% — 15461 37 3′-UTR  62% 38%15422 38 3′-UTR 153% — 15463 38 3′-UTR  91%  9% 15424 39 3′-UTR 207% —15465 39 3′-UTR  88% 12% 15426 40 3′-UTR 171% — 15467 40 3′-UTR 105% —15428 41 3′-UTR  95%  5% 15469 41 3′-UTR  96%  4% 15430 42 3′-UTR 137% —15471 42 3′-UTR 131% —

Example 3

[0121] Dose Response of Antisense Phosphorothioate OligonucleotideEffects on FAK Levels in A549 Cells

[0122] Several of the more active oligonucleotides were chosen for adose response study. A549 cells were grown, treated and processed asdescribed in Example 2, except the concentration of oligonucleotide wasvaried.

[0123] Results are shown in Table 7. Many oligonucleotides showed IC₅₀sof 50 nM or less and maximal inhibition seen was 95%. TABLE 7 DoseResponse of A549 cells to FAK Phosphorothioate Oligonucleotides SEQ ASOISIS ID Gene % mRNA % mRNA # NO: Target Dose Expression Inhibitioncontrol — — — 100.0% — 15932  3 5′-UTR  50 nM  80.3% 19.7% 15932  35′-UTR 200 nM  41.6% 58.4% 15932  3 5′-UTR 400 nM  28.3% 71.7% 15393 235′-UTR  50 nM 116.6% — 15393 23 5′-UTR 200 nM  87.8% 12.2% 15393 235′-UTR 400 nM  60.7% 39.3% 15401  8 5′-UTR  50 nM  31.9% 68.1% 15401  85′-UTR 200 nM  26.8% 73.2% 15401  8 5′-UTR 400 nM  20.4% 79.6% 15403  95′-UTR  50 nM  82.7% 17.3% 15403  9 5′-UTR 200 nM  27.8% 72.2% 15403  95′-UTR 400 nM  18.6% 81.4% 15406 30 start  50 nM  51.6% 48.4% 15406 30start 200 nN  40.5% 59.5% 15406 30 start 400 nM  39.3% 60.7% 15408 31coding  50 nM  47.7% 52.3% 15408 31 coding 200 nM  67.8% 32.2% 15408 31coding 400 nM  53.2% 46.8% 15409 12 coding  50 nM  30.1% 69.9% 15409 12coding 200 nM  29.7% 70.3% 15409 12 coding 400 nM  18.9% 81.1% 15413 14coding  50 nM  45.6% 54.4% 15413 14 coding 200 nM  21.6% 78.4% 15413 14coding 400 nM  20.6% 79.4% 15415 15 coding  50 nM  46.9% 53.1% 15415 15coding 200 nM  18.0% 82.0% 15415 15 coding 400 nM  8.0% 92.0% 15421 183′-UTR  50 nM  25.0% 75.0% 15421 18 3′-UTR 200 nM  14.8% 85.2% 15421 183′-UTR 400 nM  5.0% 95.0%

[0124] A dose response experiment on protein levels was done with twooligonucleotides. A549 cells were grown and treated as described inExample 2 except the concentration was varied as shown in Table 3. TheLIPOFECTIN^(R) to oligonucleotide ratio was maintained at 3 mg/mlLIPOFECTIN^(R) per 100 nM oligonucleotide. FAK protein levels weredetermined 48 hours after antisense treatment in whole cell lysates byanti-FAK blotting. Cells on 10cm plates were lysed with 0.5 ml modifiedRIPA lysis buffer, diluted with 0.5 ml HNTG buffer (50 mM HEPES, pH 7.4,150 mM NaCl, 0.1% Triton X-100, 10% glycerol), incubated with agarosebeads, and cleared by centrifugation. Immunoprecipitations with apolyclonal FAK antibody (Salk Institute of Biological Studies, La Jolla,Calif.; additional FAK antibodies available from Upstate BiotechnologyIncorporated, Lake Placid, N.Y.) were performed for 4 hr at 4° C.,collected on protein A (Repligen, Cambridge, Mass.) or protein G-plus(Calbiochem) agarose beads, and the precipitated protein complexes werewashed at 4° C. in Triton only lysis buffer (modified RIPA withoutsodium deoxycholate and SDS) followed by washing in HNTG buffer prior todirect analysis by SDS-PAGE. For immunoblotting, proteins weretransferred to polyvinylidene fluoride membranes (Millipore) andincubated with a 1:1000 dilution of polyclonal antibody for 2 hr at roomtemperature. Bound primary antibody was visualized by enhancedchemiluminescent detection.

[0125] Results are shown in Table 8. TABLE 8 Dose Response of A549 cellsto FAK Phosphorothioate Oligonucleotides SEQ ASO ISIS ID Gene % protein% protein # NO: Target Dose Expression Inhibition control — — — 100% —15409 12 coding  25 nM  60% 40% 15409 12 coding 100 nM  57% 43% 15409 12coding 200 nM  23% 77% 15421 18 3′-UTR  25 nM  73% 27% 15421 18 3′-UTR100 nM  34% 66% 15421 18 3′-UTR 200 nM  24% 76%

Example 4

[0126] Effect of FAK Antisense Phosphorothioate Oligonucleotides onGrowth Factor Stimulated Migration and Invasion

[0127] Integrin-regulated focal adhesion kinase (FAK) is an importantcomponent of epidermal (EGF) and platelet-drived (PDGF) growthfactor-induced motility of primary fibroblasts, smooth muscle, andadenocarcinoma cells. To measure the effect of FAK antisenseoligonucleotides on cell migration, a modified Boyden chamber(Millipore, Bedford, Mass.) assay was used (Sieg, D. J., et al., J. CellSci., 1999, 112, 2677-2691). Both membrane sides were coated with rattail collagen (5 ?g/ml in PBS, Boehringer Mannheim) for 2 hr at 37° C.,washed with PBS, and the chambers were placed into 24 well dishescontaining migration media (0.5 ml DMEM containing 0.5% BSA) with orwithout human recombinant PDGF-BB, EGF, or basic-FGF (Calbiochem, SanDiego, Calif.) at the indicated concentrations. Serum-starved A549 cells(1×10⁵ cells in 0.3 ml migration media) were added to the upper chamberand after 3 hr at 37° C., the cells on the membrane upper surface wereremoved by a cotton tip applicator, the migratory cells on the lowermembrane surface were fixed, stained (0.1% crystal violet, 0.1 M boratepH 9.0, 2% EtOH), and the dye eluted for absorbance measurements at 600nM. Individual experiments represent the average from three individualchambers. Background levels of cell migration (less than 5% of total) inthe absence of chemotaxis stimuli (0.5% BSA only) were subtracted fromall points.

[0128] Results are shown in Table 9. ISIS 17636 (SEQ ID NO. 43) is afive base mismatch control oligonucleotide for ISIS 15421 (SEQ ID NO.18). TABLE 9 Effect of FAK Antisense Phosphorothioate Oligonucleotideson EGF-Stimulated Cell Migration ISIS SEQ ID ASO Gene EGF # NO: Target(ng/ml) A₆₀₀ control — — 2.5 0.74 15421 18 3′-UTR 2.5 0.26 17636 43control 2.5 0.90 control — — 5.0 0.89 15421 18 3′-UTR 5.0 0.25 17636 43control 5.0 0.77

[0129] FAK antisense oligonucleotides were tested in an in vitroinvasion assay using an ˜1 mm MATRIGEL^(R) (Becton Dickinson, FranklinLakes, N.J.) basement membrane barrier (Albini, A., Pathol. Oncol. Res.,1998, 4, 230-241). Migration chambers were coated with the indicatedconcentration of MATRIGEL^(R), dried under laminar flow and thenrehydrated with cold serum free DMEM for 90 min on an orbital shaker.A549 cells were grown and transfected as described in Example 2. Cells(1×10⁵) were then placed onto the MATRIGEL^(R) coated membrane andallowed to invade through the MATRIGEL^(R) towards a 100 FBSchemoattractant for the indicated times. Cells that invaded through theMATRIGEL^(R) were visualized by crystal violet staining as detailed inthe migration assay. The amount of MATRIGEL^(R) was varied in the assayto show that invasion was being measured and that the migration was notserum-induced.

[0130] Results are shown in Table 10. TABLE 10 Effect of FAK AntisensePhosphorothioate Oligonucleotides on Tumor Cell Invasion ISIS SEQ ID ASOGene MATRIGEL^(R) Migration # NO: Target (μg/chamber) (A₆₀₀) control — — 0 8.3 15421 18 3′-UTR  0 2.8 17636 43 control  0 9.9 control — — 15 4.515421 18 3′-UTR 15 2.0 17636 43 control 15 4.3 control — — 26 1.6 1542118 3′-UTR 26 0.7 17636 43 control 26 1.3

Example 5

[0131] FAK Antisense Oligonucleotides in a Retinal NeovascularizationModel

[0132] FAK antisense oligonucleotides were tested in a rabbit model ofretinal neovascularization (Kimura, H., et al., Invest. Opthalmol. Vis.Sci., 1995, 36, 2110-2119). In this model, growth factors areencapsulated and injected beneath the retina.

[0133] Eight male Dutch Belt rabbits and one male Black Satin/NewZealand White Cross rabbit were used in this study. ISIS 15409 (SEQ IDNO. 12) was administered intravitreally by injection, once prior tosurgical implantation of the polymeric pellets and once during pelletimplantation. Retinal neovascularization was monitored by indirectopthalmolscopy and documented by fundus photography. Retinalneovascularization was graded on a scale from 1 to 5, with one beingnormal and five showing retinal hemorrhaging and/or detachment. Inanimals injected with saline and the growth factor containing pellets,evidence of retinal neovascularization could be detected in the firstweek and retinal hemorrhaging began by the end of the third week.Animals receiving the antisense FAK oligonucleotide showed no evidenceof retinal neovascularization over a four week period.

Example 6

[0134] Effect of FAK Antisense Phosphorothioate Pligonucleotide (ISIS15421) Alone and in Combination with 5-Flurouracil on the Viability ofMelanoma Cell Lines

[0135] Inhibition of FAK in tumor cell lines causes cell rounding, lossof adhesion, and apoptosis which suggests a role for these inhibitors inthe treatment of metastatic conditions. In these studies, an antisenseinhibitor of FAK was tested alone and in combination with thechemotherapeutic agent, 5-FU for its effects on melanoma cell lineviability. C8161 and BL human melanoma cell lines were treated with ISIS15421 (SEQ. ID. NO 18) or a control oligonucleotide, ISIS 29848, a20-mer random oligonucleotide (NNNNNNNNNNNNNNNNNNNN, wherein each N is amixture of A, C, G and T; herein incorporated as SEQ ID NO: 44) usingthe lipofectin protocol described herein. Oligonucleotides weretransfected for four hours at 300 nM in lipofectin reagent and 5-FU (200μg/mL; SIGMA) was added after the incubation for 20 hours. Cellviability was determined by the MTT assay. Loss of adhesion andapoptosis were determined by cell counting and the TUNEL assay,respectively. FAK expression was assayed by Western blot, probing withthe anti-FAK clone 4.47 antibody (Upstate Biotechnology, Lake Placid,N.Y.).

[0136] In The BL melanoma cell line, treatment with ISIS 15421 resultedin a 23% reduction in cell viability compared to control (p<0.0001).Addition of 5-FU to the antisense treated cells resulted in asignificant further reduction in cell viability (69%; p<0.0001) comparedto treatment with ISIS 15421 or 5-FU alone (4.4% reduction; p=0.15) orthe control oligonucleotide, ISIS 29848. Similar results were seen withthe C8161 cell line.

[0137] In both cell lines, reduction in cell viability was accompaniedby a proportional loss of cell adhesion and an increase in apoptosis.Western blots showed that treatment with ISIS 15421 resulted in adecrease of FAK protein expression. FAK protein levels were decreased inBL melanoma cells upon treatment with 5-FU alone and were undetectableupon treatment with the combination of ISIS 15421 and 5-FU. Thesestudies suggest that ISIS 15421, in combination with thechemotherapeutic agent 5-FU, may be a useful in the treatment ofmelanoma.

Example 7

[0138] Effect of FAK Antisense Phosphorothioate Oligonucleotide (ISIS15421) on Human Melanoma Xenograft Tumor Growth in Mice

[0139] Another model used to investigate the efficacy of antisenseoligonucleotides on tumor growth involves the use of mice transplantedwith human cancer cells or cell line tumors. In these experiments humanC8161 melanoma tumor xenografts were transplanted onto the side of nudemice with sutures or surgical staples. Mice were treated with ISIS 15421(SEQ ID NO. 18) or the control ISIS 29848 (SEQ ID NO. 44) over a 28 daytreatment course.

[0140] At the end of the timecourse, mice were sacrificed and tumorvolumes measured. Tumor volumes in the antisense treated mice weresignificantly smaller than tumor volumes in control-treated mice with noobservation of toxicity to the mice. Additionally, one third of thecontrol-treated mice had grossly evident intraperitoneal metastases,while none of the antisense-treated mice displayed such metastases.These studies suggest that antisense oligonucleotides representpotential chemotherapeutic agents in the treatment of melanoma and theprevention of tumor metastasis.

What is claimed is:
 1. An antisense compound 8 to 30 nucleobases inlength targeted to the 5′-untranslated region, translational terminationregion or 3′ untranslated region of a nucleic acid molecule encodingfocal adhesion kinase, wherein said antisense compound inhibits theexpression of said focal adhesion kinase.
 2. The antisense compound ofclaim 1 which is an antisense oligonucleotide.
 3. The antisense compoundof claim 2 wherein the antisense oligonucleotide has a sequencecomprising SEQ ID NO: 3, 4, 6, 7, 8, 9, 16, 17, 18, 20 or
 23. 4. Theantisense compound of claim 2 wherein the antisense oligonucleotidecomprises at least one modified internucleoside linkage.
 5. Theantisense compound of claim 4 wherein the modified internucleosidelinkage is a phosphorothioate linkage.
 6. The antisense compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 7. The antisense compound of claim 6 wherein themodified sugar moiety is a 2′-O-methoxyethyl moiety.
 8. The antisensecompound of claim 2 wherein the antisense oligonucleotide comprises atleast one modified nucleobase.
 9. The antisense compound of claim 8wherein the modified nucleobase is a 5-methyl cytosine.
 10. Theantisense compound of claim 2 wherein the antisense oligonucleotide is achimeric oligonucleotide.
 11. A pharmaceutical composition comprisingthe antisense compound of claim 1 and a pharmaceutically acceptablecarrier or diluent.
 12. The pharmaceutical composition of claim 11further comprising a colloidal dispersion system.
 13. The pharmaceuticalcomposition of claim 11 wherein the antisense compound is an antisenseoligonucleotide.
 14. The pharmaceutical composition of claim 11 furthercomprising a chemotherapeutic agent.
 15. The pharmaceutical compositionof claim 14 wherein the chemotherapeutic agent is 5-fluorouracil.
 16. Amethod of inhibiting the growth of a tumor in an animal comprisingadministering to said animal an effective amount of the pharmaceuticalcomposition of claim 14 .
 17. A method of inhibiting the expression offocal adhesion kinase in cells or tissues comprising contacting saidcells or tissue with the antisense compound of claim 1 so thatexpression of focal adhesion kinase is inhibited.
 18. An antisensecompound up to 30 nucleobases in length targeted to the coding region,or start site of a nucleic acid molecule encoding focal adhesion kinase,wherein said antisense compound inhibits the expression of said focaladhesion kinase and has a sequence comprising at least an 8 nucleobasicportion of SEQ ID NO: 10, 11, 12, 14, 15, 30, 31 or
 33. 19. Theantisense compound of claim 18 which is an antisense oligonucleotide.20. The antisense compound of claim 19 wherein the antisenseoligonucleotide comprises at least one modified internucleoside linkage.21. The antisense compound of claim 20 wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 22. The antisensecompound of claim 19 wherein the antisense oligonucleotide comprises atleast one modified sugar moiety.
 23. The antisense compound of claim 22wherein the modified sugar moiety is a 2′-O-methoxyethyl moiety.
 24. Theantisense compound of claim 19 wherein the antisense oligonucleotidecomprises at least one modified nucleobase.
 25. The antisense compoundof claim 24 wherein the modified nucleobase is a 5-methyl cytosine. 26.The antisense compound of claim 19 wherein the antisense oligonucleotideis a chimeric oligonucleotide.
 27. A pharmaceutical compositioncomprising the antisense compound of claim 18 and a pharmaceuticallyacceptable carrier or diluent.
 28. The pharmaceutical composition ofclaim 27 further comprising a colloidal dispersion system.
 29. Thepharmaceutical composition of claim 27 wherein the antisense compound isan antisense oligonucleotide.
 30. The pharmaceutical composition ofclaim 27 further comprising a chemotherapeutic agent.
 31. Thepharmaceutical composition of claim 30 wherein the chemotherapeuticagent is 5-fluorouracil.
 32. A method of inhibiting the growth of atumor in an animal comprising administering to said animal an effectiveamount of the pharmaceutical composition of claim 30 .
 33. A method ofinhibiting the expression of focal adhesion kinase in cells or tissuescomprising contacting said cells or tissue with the antisense compoundof claim 18 so that expression of focal adhesion kinase is inhibited.34. A method of treating an animal having a disease or conditionassociated with focal adhesion kinase comprising administering to saidanimal a therapeutically or prophylactically effective amount of anantisense compound 8 to 30 nucleobases in length targeted to a nucleicacid molecule encoding human focal adhesion kinase wherein saidantisense compound inhibits the expression of human focal adhesionkinase.
 35. The method of claim 34 wherein the disease or condition iscancer.
 36. The method of claim 35 wherein said cancer is of the breast,colon, mouth or skin.
 37. The method of claim 34 wherein said disease orcondition is an angiogenic disorder.
 38. The method of claim 37 whereinsaid angiogenic disorder is retinal neovascularization.
 39. A method ofpreventing migration of cells associated with expression of focaladhesion kinase comprising administering to said cells a therapeuticallyor prophylactically effective amount of an antisense compound 8 to 30nucleobases in length targeted to a nucleic acid molecule encoding humanfocal adhesion kinase wherein said antisense compound inhibits theexpression of human focal adhesion kinase.
 40. A method of preventingneovascularization associated with expression of focal adhesion kinasein an animal comprising administering to said animal a therapeuticallyor prophylactically effective amount of an antisense compound 8 to 30nucleobases in length targeted to a nucleic acid molecule encoding humanfocal adhesion kinase wherein said antisense compound inhibits theexpression of human focal adhesion kinase.
 41. A method of treating ananimal having a disease or condition associated with focal adhesionkinase comprising administering to said animal a therapeutically orprophylactically effective amount of an antisense compound 8 to 30nucleobases in length targeted to a nucleic acid molecule encoding humanfocal adhesion kinase in combination with a therapeutically orprophylactically effective amount of a chemotherapeutic agent.
 42. Themethod of claim 41 wherein the chemotherapeutic agent is 5-fluorouracil.43. The method of claim 41 wherein the disease or condition is cancer.44. The method of claim 43 wherein said cancer is melanoma.